Isotope enhanced ambroxol for long lasting autophagy induction

ABSTRACT

The present invention is directed to 13C and/or 2H isotope enhanced ambroxol (“isotope enhanced ambroxol”) and its use in the treatment of autophagy infections, especially mycobacterial and other infections, disease states and/or conditions of the lung, such as tuberculosis, especially including drug resistant and multiple drag resistant tuberculosis. Pharmaceutical compositions comprising isotope enhanced ambroxol, alone or in combination with an additional bioactive agent, especially rifamycin antibiotics, including an additional autophagy modulator (an agent which is active to promote or inhibit autophagy), thus being useful against, an autophagy mediated disease state and/or condition), especially an autophagy mediated disease state and/or condition which occurs in the lungs, for example, a Mycobacterium infection. Chronic Obstructive Pulmonary Disease (COPD), asthma, pulmonary fibrosis, cystic fibrosis, Sjogren&#39;s disease and lung cancer (small cell and non-small cell lung cancer, among other disease states and/or conditions, especially of the lung. Methods of treating autophagy disease states and/or conditions, especially including autophagy disease states or conditions which occur principally in the lungs of a patient represent a further embodiment of the present invention. An additional embodiment includes methods of synthesizing compounds according to the present invention as otherwise disclosed herein.

RELATED APPLICATIONS

The present application claims the benefit of priority of U.S.provisional application Ser. No. 62/455,822 filed Feb. 7, 2017 ofidentical title, the entire contents of said application beingincorporated herein.

FIELD OF THE INVENTION

The present invention is directed to ¹³C and or ²H isotope enhancedambroxol (“isotope enhanced ambroxol”) and its use in the treatment ofautophagy infections, especially mycobacterial infections of the lung,such as tuberculosis, especially including drug resistant and multipledrug resistant tuberculosis, and also including a range of rifampinsensitive lung infections such as pneumonias caused by methicillinresistant Staphyloccus aureus (MRSA) carbapenem-resistant Acinetobacterbaumanii, and inhalational anthrax (caused by Bacillus anthracis).Pharmaceutical compositions comprising isotope enhanced ambroxol, aloneor in combination with an additional bioactive agent, including anadditional autophagy modulator (an agent which is active to promote orinhibit autophagy), thus being useful against an autophagy mediateddisease state and/or condition), especially an autophagy mediateddisease state and/or condition which occurs in the lungs, for example, aMycobacterium infection, a non-mycobacterium lung infection, ChronicObstructive Pulmonary Disease (COPD), asthma, pulmonary fibrosis, cysticfibrosis, Sjogren's disease and lung cancer (especially small cell andnon-small cell lung cancer). Methods of treating autophagy diseasestates and/or conditions, especially including autophagy disease statesor conditions which occur principally in the lungs of a patientrepresent a further embodiment of the present invention. An additionalembodiment includes methods of synthesizing compounds according to thepresent invention as otherwise disclosed herein.

BACKGROUND AND OVERVIEW OF THE INVENTION

Unpublished data from the inventors evidences that Ambroxol is apowerful inducer of autophagy. However, its plasma half-life in humansis only 3.72 hours (1) meaning multiple doses daily need to be given inorder to provide an effective concentration. Its major route ofelimination is via urinary excretion of a glucuronide (2) conjugate, soany other factors are not predicted to alter metabolism or tissuepharmacokinetics or dynamics.

Ambroxol's major metabolism by human microsomes was shown to be bycyp3A4 mediated oxidation to dibromoanthranilic acid (DBAA).(3) SeeFIG. 1. Thus, any potential use of ambroxol in treating diseasesautophagy-mediated disease states and/or conditions like tuberculosis(TB) may be greatly compromised by the cyp3A4 inducing nature of many TBmedicines such as rifampin. Alternatively, the use of cyp3A4 inhibitingmedications with ambroxol may also lead to greater than desired levelsof ambroxol because it is no longer being metabolized by cyp3A4. Thus,unfortunately, the inherent susceptibility of ambroxol to cyp3A4 willmake it difficult to dose in humans in diseases such as TB treated withrifampin or other anti-TB agents, or when cyp3A4 inhibiting compoundsare given.

Furthermore, ambroxol displays lung concentrating effects that willpotentiate its effects in lung diseases such as tuberculosis. It israrely acknowledged that lung tissues express significant cyp3A4, aswell as the more commonly understood liver expression, and cyp3A4 isfound in human lungs in sites including bronchial glands, bronchiolarcolumnar and terminal epithelium, type II alveolar epithelium, andalveolar macrophages.(4) Therefore, even if ambroxol is given in inhaledforms, it will inherently and unexpectedly be metabolized into inactiveforms by lung tissue cyp3A4 limiting lung exposure levels to ambroxol.

Brief Description of the Invention

In one embodiment, the inventors disclose isotope enhanced forms ofambroxol (isotope enhanced ambroxol 1 through 4—see attached FIG. 1)that will resist cyp3A4 metabolism. This series of compounds will allowmore optimal use of ambroxol, especially orally or inhaled, and will beinherently resistant to unexpected alterations in plasma or lung tissuelevels from cyp3A4 presence, induction or inhibition.

According to the present invention, the following isotopes ¹³C and/or ²Hisotopes of ambroxol are provided. Thus, a first embodiment is directedto isotopically labeled compounds according to the chemical formula:

or a pharmaceutically acceptable salt, stereoisomer (including adiastereomer and/or enantiomer), solvate or polymorph thereof.

In another embodiment, the present invention is directed topharmaceutical compositions comprising an effective amount of at leastone isotopically labeled ambroxol compound as described above, incombination with a pharmaceutically acceptable additive, carrier and/orexcipient. Pharmaceutical compositions described above may be optionallycombined with at least one additional bioactive agent, preferably ananticancer agent (preferably an anticancer agent effective to treat lungcancer, including small cell lung cancer and non-small cell lung cancersuch as Cisplatin, Carboplatin, Paclitaxel (Taxol), Albumin-boundpaclitaxel (nab-paclitaxel, Abraxane), Docetaxel (Taxotere), Gemcitabine(Gemzar), Vinorelbine (Navelbine), Irinotecan (Camptosar), Etoposide(VP-16), Vinblastine, Pemetrexed (Alimta) and mixtures thereof), or anagent which is useful in the treatment of a mycobacterial infection,especially including a tuberculosis infection or another autophagymediated disease. In certain embodiments, the additional bioactive agentis an anti-tuberculosis agents such as ethambutol, isoniazid,ethionamide, prothionamide, pyrazinamide, rifampacin or mixturesthereof. In other embodiments, the additional bioactive agent is anautophagy modulator as otherwise described herein. In still otherembodiments, the additional bioactive agent may include both ananti-tuberculosis agent and/or an additional autophagy modulator asdescribed herein. In still other embodiments the bioactive agent is orincludes rifampin and other rifamycin derivatives such as rifabutin,rifamixin and rifapentine are vitally important drugs in regimens fornon-tuberculous mycobacteria lung (NTM) infections such as Mycobacteriumavium complex (MAC) and M. kansasii.

In another embodiment, the present invention is directed to a method fortreating an autophagy-mediated disease state or condition in a patient,especially including an autophagy-mediated disease state or conditionprincipally localized in the lungs of a patient, comprisingadministering an effective amount of at least one isotopically labeledambroxol compound to a patient in need, optimally in combination with atleast one additional bioactive agent, which bioactive agent may includean anti-tuberculosis agent and/or an autophagy modulator. While anynumber of autophagy mediated disease states and/or conditions may betreated using compounds and optional agents according to the presentinvention, in preferred embodiments, the present methods are directed tothe treatment of diseases which are principally found in the lungs of apatient. The disease states and/or conditions of the lung which may betreated pursuant to the present invention include, for example, aMycobacterium infection (especially including tuberculosis) butincluding other infections such as tuberculous and non-tuberculousmycobacteria (such as M. kansasii, M. avium and M. abscessus),non-mycobacterial lung infections, such as pneumonias caused bymethicillin resistant Staphylococcus aureus (MRSA), carbapenem-resistantAcinebacter baumanii, and inhalational anthrax, Chronic ObstructivePulmonary Disease (COPD), asthma, pulmonary fibrosis, cystic fibrosis,Sjogren's disease and lung cancer (small cell and non-small cell lungcancer). In still other embodiments the bioactive agent is or includesrifampin and other rifamycin derivatives such as rifabutin, rifamixinand rifapentine which are vitally important drugs in regimens fornon-tuberculous mycobacteria lung (NTM) infections such as Mycobacteriumavium complex (MAC), M. kansasii and M. abscessus. For TB, standard‘short’ course therapy includes 6 months therapy with rifampin. In NTMtherapy even longer treatment courses are required. For MAC, the drugtreatment cessation is only recommended after 12 months of sputumculture negativity (SCN), so treatments greatly in excess of 1 year arecommon. The recommendations for M. kansasii are also for 20 monthstherapy with at least 12 months of therapy after SCN is achieved. Theseare very long treatment courses compared to most bacterial disease, andtheir treatment can be meaningfully improved and therapy times shortenedby rifamycin derivatives such as rifampin, rifabutin, rifamixin andrifapentine (preferably rifampin) potentiation by the agents describedherein.

In another embodiment, isotopically enhanced ambroxol can potentiaterifampin and other rifamycin derivatives such as rifabutin, rifamixinand rifapentine, alone or in mixtures thereof for treatingnon-mycobacterial lung infections, such as pneumonias caused bymethicillin resistant Staphylococcus aureus (MRSA), carbapenem-resistantAcinetobacter baumanii, and inhalational anthrax, the method comprisingadministering an effective amount of one or more of the above agents toa patient in need of therapy thereof.

In another embodiment, the present invention is directed to a method ofimproving the pharmacokinetics and/or bioavailability of ambroxol in apatient after administration comprising administering an isotopicallylabeled ambroxol compound as described herein to a patient in need.

In yet another embodiment, the present invention is directed to a methodof synthesizing isotopically labeled ambroxol, the method comprising (asdepicted in FIG. 2 hereof) reacting 4 amino cyclohexanol of depictedconfiguration (FIG. 2) with an isotopically labeled dibromo aminobenzaldehyde to provide the isotopically labeled imine (FIG. 2) which isfurther reduced to provide one or more of the isotopically labeledcompounds according to the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the various isotopically labeled ambroxol derivativesaccording to the present invention. Compounds which are exhibited inFIG. 1 include trans-4-[(2-amino-3,5-dibromobenzyl)amino]cyclhexanol(ambroxol), trans-4-{[(2-amino-3,5-dibromobenzyl)(²H₁,¹H₁)methyl]amino]cyclhexanol (isotope enhanced ambroxol 1),trans-4-{[(2-amino-3,5-dibromobenzyl)(²H₂)methyl]amino]cyclhexanol(isotope enhanced ambroxol 2),trans-4-{[(2-amino-3,5-dibromobenzyl)(²H₂, ¹³C)methyl]amino]cyclhexanol(isotope enhanced ambroxol 3) andtrans-4-{[(2-amino-3,5-dibromobenzyl)(²H₁, ¹H₁,¹³C)methyl]amino]cyclhexanoI (isotope enhanced ambroxol 4) The isotopicambroxol compounds are resistant to Cyp3A4 metabolism. This figure showsambroxol and the isotopic forms 1-4 and how they will resist cyp3A4metabolism.

FIG. 2, scheme 1, provides a synthetic scheme for producingisotopically-labeled ambroxol compounds according to the presentinvention. In this method an enantiomerically enriched1-hydroxy-4-aminocyclohexane is reacted with an isotopically enricheddibromoaminobenzaldehyde as indicated. The imine which is formed is thensubjected to reduction using an isotopically labeled reducing agent toprovide the final isotopically labeled ambrosol. Various iterations ofthe compounds according to the present invention are readily produced byvarying the atom which is isotopically labeled in thedibromoaminobenzaldehyde compound or the reducing agent.

FIG. 3 shows the dose response of wild type cells. This figure clearlyshows that even concentrations as low as 6.25 μM had a positive effectson autophagy (t test v. control p=0.025). The data show that across therange of ambroxol concentrations from 0 to 200 uM, higher ambroxolconcentrations result in higher induction of autophagy.

FIG. 4 shows results from the same dose response assay used to generatethe data in FIG. 3, but with ATG5 knockout cells, rather than wild typecells. The data shown in this FIG. 4 evidences that even concentrationsof ambroxol as low as 6.25 uM gave appreciable increases in autophagy (ttest v. control p=0.0008). Again a clear dose response as observed.

FIGS. 5 and 6 show ambroxol induction of TFEB nuclear translocation togauge activity of ambroxol as an inducer of autophagy. A sigmoidal fitof both data sets indicated 50% of maximal autophagy induced by ambroxolwas found at about 50 uM. Again significant increases were observed at25 uM. Unlike the LC3 marker of autophagy, ambroxol always gave lesseffect than pp242. It is clear in all cases that increasing levels ofambroxol will increase autophagic responses.

DETAILED DESCRIPTION OF THE INVENTION

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” include plural referents unlessexpressly and unequivocally limited to one referent. Thus, for example,reference to “a compound” includes two or more different compound. Asused herein, the term “include” and its grammatical variants areintended to be non-limiting, such that recitation of items in a list isnot to the exclusion of other like items that can be substituted orother items that can be added to the listed items. It is noted that whena range is described in the specification, every single integer fallingwithin the range is also included within the range, unless the contextof the use of the range specifically excludes an integer within thatrange.

The term “compound” or “agent”, as used herein, unless otherwiseindicated, refers to any specific chemical compound disclosed herein,especially including ambroxol or a derivative thereof or other autophagymodulator and includes tautomers, regioisomers, geometric isomers asapplicable, and also where applicable, optical isomers (e.g.enantiomers) thereof, as well as pharmaceutically acceptable saltsthereof. Within its use in context, the term compound generally refersto a single compound, but also may include other compounds such asstereoisomers, regioisomers and/or optical isomers (including racemicmixtures) as well as specific enantiomers or enantiomerically enrichedmixtures of disclosed compounds as well as diastereomers and epimers,where applicable in context. The term also refers, in context to prodrugforms of compounds which have been modified to facilitate theadministration and delivery of compounds to a site of activity.

The term “patient” or “subject” is used throughout the specificationwithin context to describe an animal, generally a mammal, including adomesticated mammal including a farm animal (dog, cat, horse, cow, pig,sheep, goat, etc.) and preferably a human, to whom treatment, includingprophylactic treatment (prophylaxis), with the methods and compositionsaccording to the present invention is provided. For treatment of thoseconditions or disease states which are specific for a specific animalsuch as a human patient, the term patient refers to that specificanimal, often a human.

The terms “effective” or “pharmaceutically effective” are used herein,unless otherwise indicated, to describe an amount of a compound orcomposition which, in context, is used to produce or affect an intendedresult, usually the modulation of autophagy within the context of aparticular treatment in the lungs of a patient or subject.

The terms “treat”, “treating”, and “treatment”, as used herein, refer toany action providing a benefit to a patient at risk for or afflicted byan autophagy mediated disease state or condition as otherwise describedherein. The benefit may be in curing the disease state or condition,inhibition its progression, or ameliorating, lessening or suppressingone or more symptom of an autophagy mediated disease state or condition.Treatment, as used herein, encompasses both prophylactic and therapeutictreatment.

As used herein, the term “autophagy mediated disease state or condition”or an “autophagy mediated lung disease” refers to a disease state orcondition that results from disruption in autophagy or cellularself-digestion and occurs in the lung of a patient or subject. Autophagyis a cellular pathway involved in protein and organelle degradation, andhas a large number of connections to human disease. Autophagicdysfunction is associated with cancer, neurodegeneration, microbialinfection and ageing, among numerous other disease states and/orconditions. Although autophagy plays a principal role as a protectiveprocess for the cell, it also plays a role in cell death. Disease statesand/or conditions which are mediated through autophagy (which refers tothe fact that the disease state or condition may manifest itself as afunction of the increase or decrease in autophagy in the patient orsubject to be treated and treatment requires administration of aninhibitor or agonist of autophagy in the patient or subject).

Disease states and/or conditions which are mediated through autophagy(which refers to the fact that the disease state or condition maymanifest itself as a function of the increase or decrease in autophagyin the patient or subject to be treated and treatment requiresadministration of an inhibitor or agonist of autophagy in the patient orsubject) include, for example, cancer, including metastasis of cancer,lysosomal storage diseases (discussed herein below), neurodegeneration(including, for example, Alzheimer's disease, Parkinson's disease,Huntington's disease; other ataxias), immune response (T cellmaturation, B cell and T cell homeostasis, counters damaginginflammation) and chronic inflammatory diseases (may promote excessivecytokines when autophagy is defective), including, for example,inflammatory bowel disease, including Crohn's disease, rheumatoidarthritis, lupus, multiple sclerosis, chronic obstructive pulmonarydisease/COPD, pulmonary fibrosis, cystic fibrosis, Sjogren's disease;hyperglycemic disorders, diabetes (I and II), affecting lipid metabolismislet function and/or structure, excessive autophagy may lead topancreatic β-cell death and related hyperglycemic disorders, includingsevere insulin resistance, hyperinsulinemia, insulin-resistant diabetes(e.g. Mendenhall's Syndrome, Werner Syndrome, leprechaunism, andlipoatrophic diabetes) and dyslipidemia (e.g. hyperlipidemia asexpressed by obese subjects, elevated low-density lipoprotein (LDL),depressed high-density lipoprotein (HDL), and elevated triglycerides andmetabolic syndrome, liver disease (excessive autophagic removal ofcellular entities-endoplasmic reticulum), renal disease (apoptosis inplaques, glomerular disease), cardiovascular disease (especiallyincluding ischemia, stroke, pressure overload and complications duringreperfusion), muscle degeneration and atrophy, symptoms of aging(including amelioration or the delay in onset or severity or frequencyof aging-related symptoms and chronic conditions including muscleatrophy, frailty, metabolic disorders, low grade inflammation,atherosclerosis and associated conditions such as cardiac andneurological both central and peripheral manifestations includingstroke, age-associated dementia and sporadic form of Alzheimer'sdisease, pre-cancerous states, and psychiatric conditions includingdepression), stroke and spinal cord injury, arteriosclerosis, infectiousdiseases (microbial infections, removes microbes, provides a protectiveinflammatory response to microbial products, limits adaptation ofautophagy of host by microbe for enhancement of microbial growth,regulation of innate immunity) including bacterial, fungal, cellular andviral (including secondary disease states or conditions associated withinfectious diseases), including AIDS and tuberculosis, among others,development (including erythrocyte differentiation),embryogenesis/fertility/infertility (embryo implantation and neonatesurvival after termination of transplacental supply of nutrients,removal of dead cells during programmed cell death) and ageing(increased autophagy leads to the removal of damaged organelles oraggregated macromolecules to increase health and prolong life, butincreased levels of autophagy in children/young adults may lead tomuscle and organ wasting resulting in ageing/progeria).

The term “lysosomal storage disorder” refers to disease state orcondition that results from a defect in lysosomal storage. These diseasestates or conditions generally occur when the lysosome malfunctions.Lysosomal storage disorders are caused by lysosomal dysfunction usuallyas a consequence of deficiency of a single enzyme required for themetabolism of lipids, glycoproteins or mucopolysaccharides. Theincidence of lysosomal storage disorder (collectively) occurs at anincidence of about 1:5,000-1:10,000. The lysosome is commonly referredto as the cell's recycling center because it processes unwanted materialinto substances that the cell can utilize. Lysosomes break down thisunwanted matter via high specialized enzymes. Lysosomal disordersgenerally are triggered when a particular enzyme exists in too small anamount or is missing altogether. When this happens, substancesaccumulate in the cell. In other words, when the lysosome doesn'tfunction normally, excess products destined for breakdown and recyclingare stored in the cell. Lysosomal storage disorders are geneticdiseases, but these may be treated using autophagy modulators(autostatins) as described herein. All of these diseases share a commonbiochemical characteristic, i.e. that all lysosomal disorders originatefrom an abnormal accumulation of substances inside the lysosome.Lysosomal storage diseases mostly affect children who often die as aconsequence at an early stage of life, many within a few months or yearsof birth. Many other children die of this disease following years ofsuffering from various symptoms of their particular disorder.

Examples of lysosomal storage diseases include, for example, activatordeficiency/GM2 gangliosidosis, alpha-matmosidosis,aspartylglucoaminuria, cholesteryl ester storage disease, chronichexosaminidase A deficiency, cystinosis, Danon disease, Fabry disease,Farber disease, fucosidosis, galactosialidosis, Gaucher Disease (TypesI, II and III), GM? Ganliosidosis, including infantile, lateinfantile/juvenile and adult/chronic), Hunter syndrome (MPS II), I-Celldisease/Mucolipidosis II, Infantile Free Sialic Acid Storage Disease(ISSD), Juvenile Hexosaminidase A Deficiency, Krabbe disease, Lysosomalacid lipase deficiency, Metachromatic Leukodystrophy, Hurler syndrome,Scheie syndrome, Hurler-Scheie syndrome, Sanfilippo syndrome, MorquioType A and B, Maroteaux-Lamy, Sly syndrome, mucolipidosis, multiplesulfate deficiency, Niemann-Pick disease, Neuronal ceroidlipofuscinoses, CLN6 disease, Jansky-Bielschowsky disease, Pompedisease, pycnodysostosis, Sandhoff disease, Schindler disease, Tay-Sachsand Wolman disease, among others.

In preferred aspects, isotopically labeled ambroxol is used often totreat autophagy-mediated lung diseases, such as Mycobacterium infectionsincluding non-tuberculosis and tuberculosis, Chronic ObstructivePulmonary Disease (COPD), asthma, pulmonary fibrosis, cystic fibrosis,Sjogren's disease and lung cancer (small cell and non-small cell lungcancer). Isotopically enhanced ambroxol can also potentiate rifampin andother rifamycin derivatives such as rifabutin, rifamixin andrifapentine, alone or in mixtures thereof for treating non-mycobacteriallung infections, such as pneumonias caused by methicillin resistant.Staphylococcus aureus (MRSA), carbapenem-resistant Acinetobacterbaumanii, and inhalational anthrax.

Additional autophagy modulators (which may be coadminstered withisotopically labeled ambroxol) also include Astemizole, Chrysophanol,Emetine, Chlorosalicylanilide, Oxiconazole, Sibutramine, Proadifen,Dihydroergotamine tartrate, Terfenadine, Triflupromazine, Amiodarone,Saponin Vinblastine, Tannic acid, Fenticlor, Pizotyline malate,Piperacetazine, Oxyphencyclimine, Glyburide, Hydroxychloroquine,Methotrimeprazine, Mepartricin, Thiamylal Sodium Triclocarban,Diphenidol, Karanjin, Clovanediol diacetate, Nerolidol, Fluoxetine,Helenine, Dehydroabietamide, Dibutyl Phthalate,18-aminoabieta-8,11,13-triene sulfate, Podophyllin acetate, Berbamine,Rotenone, Rubescensin A, Morin, Pyrromycin, Pomiferin, Gardenin A,alpha-mangostin, Avocadene, Butylated hydroxytoluene, Physcion,Tetrandrine, Malathion, Isoliquiritigenin, Clofoctol, Isoreserpine,4,4′-dimethoxydalbergione and 4-methyldaphnetin, and mixtures thereof.Other agents which may be coadministered with ambroxol, includebenzethonium, niclosamide, monensin, bromperidol, levobunolol,dehydroisoandosterone 3-acetate, flubendazole, sertraline, tamoxifen,reserpine, hexachlorophene, dipyridamole, harmaline, prazosin,lidoflazine, thiethylperazine, dextromethorphan, desipramine,mebendazole, canrenone, chlorprothixene, maprotiline,homochlorcyclizine, loperamide, nicardipine, dexfenfluramine,nilvadipine, dosulepin, biperiden, denaionium, etomidate, toremifene,tomoxetine, clorgyline, zotepine, beta-escin, tridihexethyl,ceftazidime, methoxy-6-harmalan, melengestrol, albendazole, rimantadine,chlorpromazine, pergolide, cloperastine, prednicarbate, haloperidol,clotrimazole, nitrofural, iopanoic acid, naftopidil, Methimazole,Trimeprazine, Ethoxyquin, Clocortolone, Doxycycline, Pirlindolemesylate, Doxazosin, Deptropine, Nocodazole, Scopolamine, Oxybenzone,Halcinonide, Oxybutynin, Miconazole, Clomipramine, Cyproheptadine,Doxepin, Dyclonine, Salbutamol, Flavoxate, Amoxapine, Fenofibrate,Pimethixene or a pharmaceutically acceptable salt, alternative salt andmixtures thereof. One or more of these agents may be formulated ininhalation/pulmonary dosage form, but often these agents are simplycoadministered with ambroxol via other routes of administration (ie,oral, parenteral, etc.).

Other agents which may be co-administered with ambroxol includeanti-tuberculosis agents, for example ethambutol, isoniazid,ethionamide, propionamide, pyrazinamide, rifampicin, aminosalicylicacid, bedaquiline, rifabutin, rifapentine, capreomycin, cycloserine andmixtures thereof. The use of ethambutol, isoniazid, ethionamide,propionamide, pyrazinamide, rifampicin and mixtures thereof arepreferred.

The term “co-administration” or “combination therapy” is used todescribe a therapy in which at least two active compounds in effectiveamounts are used to treat an autophagy mediated disease state orcondition as otherwise described herein, either at the same time orwithin dosing or administration schedules defined further herein orascertainable by those of ordinary skill in the art. The two compoundsmay be administered in the same composition. Although the termco-administration preferably includes the administration of two activecompounds to the patient at the same time, it is not necessary that thecompounds be administered to the patient at the same time, althougheffective amounts of the individual compounds will be present in thepatient at the same time. In addition, in certain embodiments,co-administration will refer to the fact that two compounds areadministered at significantly different times, but the effects of thetwo compounds are present at the same time. Thus, the termco-administration includes an administration where ambroxol iscoadministered with at least one additional active agent (includinganother autophagy modulator) at approximately the same time(contemporaneously), or from about one to several minutes to about 24hours or more before or after the other bioactive agent is administered.The additional bioactive agent may be any bioactive agent, but is oftenan additional autophagy mediated compound which serves to enhance theactivity of isotopically labeled ambroxol or one of its derivatives.

The present invention includes compositions comprising thepharmaceutically acceptable salt. i.e., the acid or base addition saltsof compounds of the present invention and their derivatives. The acidswhich may be used to prepare the pharmceutically acceptable acidaddition salts of the aforementioned base compounds useful in thisinvention are those which form non-toxic acid addition salts, i.e.,salts containing pharmacologically acceptable anions, such as thehydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate,phosphate, acid phosphate, acetate, lactate, citrate, acid citrate,tartrate, bitartrate, succinate, maleate, fumarate, gluconate,saccharate, benzoate, methanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate and pamoate [i.e.,1,1′-methylene-bis-(2-hydroxy-3 naphthoate)]salts, among others.

Pharmaceutically acceptable base addition salts may also be used toproduce pharmaceutically acceptable salt forms of the compoundsaccording to the present invention. The chemical bases that may be usedas reagents to prepare pharmaceutically acceptable base salts of thepresent compounds that are acidic in nature are those that formnon-toxic base salts with such compounds. Such non-toxic base saltsinclude, but are not limited to those derived from suchpharmacologically acceptable cations such as alkali metal cations (eg.,potassium and sodium) and alkaline earth metal cations (e, calcium andmagnesium), ammonium or water-soluble amine addition salts such asN-methylglucamine-(meglumine), and the lower alkanolammonium and otherbase salts of pharmaceutically acceptable organic amines, among others.

As indicated, the pharmaceutical composition may comprise, in additionto at least one isotopically labeled ambroxol and optional bioactiveagent, including an autophagy modulator, a pharmaceutically acceptableexcipient, additive or inert carrier. The pharmaceutically acceptableexcipient, additive or inert carrier may be in a form chosen from asolid, semi-solid, and liquid. The pharmaceutically acceptable excipientor additive may be chosen from a starch, crystalline cellulose, sodiumstarch glycolate, polyvinylpyrolidone, polyvinylpolypyrolidone, sodiumacetate, magnesium stearate, sodium laurylsulfate, sucrose, gelatin,silicic acid, polyethylene glycol, water, alcohol, propylene glycol,vegetable oil, corn oil, peanut oil, olive oil, surfactants, lubricants,disintegrating agents, preservative agents, flavoring agents, pigments,and other conventional additives. The pharmaceutical composition may beformulated by admixing the active with a pharmaceutically acceptableexcipient or additive.

The pharmaceutical composition may be in a form chosen from sterileisotonic aqueous solutions, pills, drops, pastes, cream, spray(including aerosols), capsules, tablets, sugar coating tablets,granules, suppositories, liquid, lotion, suspension, emulsion, ointment,gel, and the like. Administration route may be chosen from subcutaneous,intravenous, intestinal, parenteral, oral, buccal, nasal, intramuscular,transcutaneous, transdermal, intranasal, intratracheal, intrapulmonary,intraperitoneal, and topical. The pharmaceutical compositions may beimmediate release, sustained/controlled release, or a combination ofimmediate release and sustained/controlled release depending upon thecompound(s) to be delivered, the compound(s), if any, to becoadministered, as well as the disease state and/or condition to betreated with the pharmaceutical composition. A pharmaceuticalcomposition may be formulated with differing compartments or layers inorder to facilitate effective administration of any variety consistentwith good pharmaceutical practice. Oral and inhalation dosage forms arepreferred, especially for the treatment of disease states and/orconditions which are localized principally in the lungs of a patient.

The subject or patient may be chosen from, for example, a human, amammal such as domesticated animal, or other animal. The subject mayhave one or more of the disease states, conditions or symptomsassociated with autophagy as otherwise described herein.

The compounds according to the present invention may be administered inan effective amount to treat or reduce the likelihood of anautophagy-mediated disease and/or condition as well one or more symptomsassociated with the disease state or condition. One of ordinary skill inthe art would be readily able to determine an effective amount of activeingredient by taking into consideration several variables including, butnot limited to, the animal subject, age, sex, weight, site of thedisease state or condition in the patient, previous medical history,other medications, etc.

For example, the dose of an active ingredient which is useful in thetreatment of an autophagy mediated disease state, condition and/orsymptom for a human patient is that which is an effective amount and mayrange from as little as 50-100 μg or even less to at least about 500 mgor more (up to several grams or more) per day, which may be administeredin a manner consistent with the delivery of the drug and the diseasestate or condition to be treated. In the case of oral administration,active is generally administered from one to four times or more daily.Transdermal patches or other topical administration may administer drugscontinuously, one or more times a day or less frequently than daily,depending upon the absorptivity of the active and delivery to thepatient's skin. Of course, in certain instances where parenteraladministration represents a favorable treatment option, intramuscularadministration or slow IV drip may be used to administer active. Theamount of active ingredient which is administered to a human patientpreferably ranges from about 0.05 mg/kg to about 25 mg/kg, about 0.1mg/kg to about 10 mg/kg, about 0.25 mg/kg to about 7.5 mg/kg., about1.25 to about 5.7 mg/kg.

The dose of a compound according to the present invention may beadministered at the first signs of the onset of an autophagy mediateddisease state, condition or symptom. For example, the dose may beadministered for the purpose of lung or heart function and/or treatingor reducing the likelihood of any one or more of the disease states orconditions which become manifest during an inflammation-associatedmetabolic disorder or tuberculosis or associated disease states orconditions, including pain, high blood pressure, renal failure, or lungfailure. The dose of active ingredient may be administered at the firstsign of relevant symptoms prior to diagnosis, but in anticipation of thedisease or disorder or in anticipation of decreased bodily function orany one or more of the other symptoms or secondary disease states orconditions associated with an autophagy mediated disorder to condition.

In preferred aspects of the invention, isotopically labeled ambroxol isadministered to the patient or subject often by oral administration oreven more often to the lungs of the patient or subject often viapulmonary administration, including intratracheal administration. Thepharmaceutical composition of the invention for pulmonary administrationis usually used as an inhalant. The composition can be formed into drypowder inhalants, inhalant suspensions, inhalant solutions, encapsulatedinhalants and like known forms of inhalants as described herein below.Such forms of inhalants can be prepared by filling the pharmaceuticalcomposition of the invention into an appropriate inhaler such as ametered-dose inhaler, dry powder inhaler, atomizer bottle, nebulizeretc. before use. Of the above forms of inhalants, powder inhalants maybe preferable, although nebulized suspensions and metered dose inhalersmay also be used effectively.

When the pharmaceutical composition of the invention is used in the formof a powder, the mean particle diameter of the powder is not especiallylimited but, in view of the residence of the particles in the lungs, ispreferable that the particles fall within the range of about 0.01 to 20μm, often 0.05 to about 5 μm (to target both macrophages and lungepithelial cells), often about 0.05 to about 0.25 μm (to target lungepithelial cells), often about 1 to 3 μm (to target lung macrophages)and particularly about 1 to 5 μm, more often about 1 to 2 μm (microns).Although the particle size distribution of the powder pharmaceuticalcomposition of the invention is not particularly limited, it ispreferable that particles having a size of about 25 μm or more accountfor not more than about 5% of the particles, and preferably, 1% or lessto maximize delivery into the lungs of the subject.

The pharmaceutical composition in the form of a powder of the inventioncan be produced by, for example, using the drying-micronization method,the spray drying method and standard pharmaceutical methodology wellknown in the art.

By way of example without limitation, according to thedrying-pulverization method, the pharmaceutical composition in the formof a powder can be prepared drying an aqueous solution (or aqueousdispersion) containing the compound or mixtures with other active agentsthereof and excipients which provide for immediate release in pulmonarytissue and microparticulating the dried product. Stated morespecifically, after dissolving (or dispersing) a pharmaceuticallyacceptable carrier, additive or excipient in an aqueous medium,compounds according to the present invention in effective amounts areadded and dissolved (or dispersed) by stirring using a homogenizer, etc.to give an aqueous solution (or aqueous dispersion). The aqueous mediummay be water alone or a mixture of water and a lower alcohol. Examplesof usable lower alcohols include methanol, ethanol, 1-propanol,2-propanol and like water-miscible alcohols. Ethanol is particularlypreferable. After the obtained aqueous solution (or aqueous dispersion)is dried by blower, lyophilization, etc., the resulting product ispulverized or microparticulated into fine particles using jet mills,ball mills or like devices to give a powder having the above meanparticle diameter. If necessary, additives as mentioned above may beadded in any of the above steps.

According to the spray-drying method, the pharmaceutical composition inthe form of a powder of the invention can be prepared, for example, byspray-drying an aqueous solution (or aqueous dispersion) containingambroxol and/or one or more ambroxol derivatives and optionally one ormore additional agents such as an additional autophagy modulator asdescribed herein and/or one or more anti-tuberculosis agent andexcipients, additives and/or carriers for microparticulation. Theaqueous solution (or aqueous dispersion) can be prepared following theprocedure of the above drying-micronization method. The spray-dryingprocess can be performed using a known method, thereby giving a powderypharmaceutical composition in the form of globular particles with theabove-mentioned mean particle diameter.

The inhalant suspensions, inhalant solutions, encapsulated inhalants,etc. can also be prepared using the pharmaceutical composition in theform of a powder produced by the drying-micronization method, thespray-drying method and the like, or by using a carrier, additive orexcipient and isoniazid, urea or mixtures thereof that can beadministered via the lungs, according to known preparation methods.

Furthermore, the inhalant comprising the pharmaceutical composition ofthe invention is preferably used as an aerosol. The aerosol can beprepared, for example, by filling the pharmaceutical composition of theinvention and a propellant into an aerosol container. If necessary,dispersants, solvents and the like may be added. The aerosols may beprepared as 2-phase systems, 3-phase systems and diaphragm systems(double containers). The aerosol can be used in any form of a powder,suspension, solution or the like.

Examples of usable propellants include liquefied gas propellants,compressed gases and the like. Usable liquefied gas propellants include,for example, fluorinated hydrocarbons (e.g., CFC substitutes such asHCFC-22, HCFC-123, HFC-134a, HFC-227 and the like), liquefied petroleum,dimethyl ether and the like. Usable compressed gases include, forexample, soluble gases (e.g., carbon dioxide, nitric oxide), insolublegases (e.g., nitrogen) and the like.

The dispersant and solvent may be suitably selected from the additivesmentioned above. The aerosol can be prepared, for example, by a known2-step method comprising the step of preparing the composition of theinvention and the step of filling and sealing the composition andpropellant into the aerosol container.

As a preferred embodiment of the aerosol according to the invention, thefollowing aerosol can be mentioned: Examples of the compounds to be usedinclude isotopically labeled compound alone or in mixtures with othercompounds according to the present invention or with otheranti-Mycobacterial agents. As propellants, fluorinated hydrocarbons suchas HFC-134a, HFC-227 and like CFC substitutes are preferable. Examplesof usable solvents include water, ethanol, 2-propanol and the like.Water and ethanol are particularly preferable. In particular, a weightratio of water to ethanol in the range of about 0:1 to 10:1 may be used.

The aerosol of the invention contains excipient in an amount rangingfrom about 0.01 to about 10⁴ wt. % (preferably about 0.1 to 10³ wt. %),propellant in an amount of about 10² to 10⁷ wt. % (preferably about 10³to 10⁶ wt. %), solvent in an amount of about 0 to 10⁶ wt. % (preferablyabout 10 to 10⁵ wt. %), and dispersant in an amount of 0 to 10³ wt. %(preferably about 0.01 to 10² wt. %), relative to the weight of compoundaccording to the present invention which is included in the finalcomposition.

The pharmaceutical compositions of the invention are safe and effectivefor use in the therapeutic methods according to the present invention.Although the dosage of the composition of the invention may varydepending on the type of active substance administered (e.g.isotopically labeled ambroxol and optionally an additional autophagymodulating agent and/or anti-tuberculosis agent such as ethambutol,isoniazid, ethionamide, prothionamide, pyrazinamide, rifampacin,mixtures thereof and optional additional anti-tuberculosis agents) aswell as the nature (size, weight, etc.) of the subject to be diagnosed,the composition is administered in an amount effective for treating anyone or more of the lung diseases mediated through autophagy. Forexample, the composition is preferably administered such that the activeingredient (isotopically labeled ambroxol and an optional agent) can begiven to a human adult in a dose of at least about 0.5-10 mg, about 1mg, to about 15 mg., about 5 mg, to about 20 mg., at least about 25 mg,at least about 50 mg, at least about 60 mg, at least about 75 mg., atleast about 100 mg, at least about 150 mg, at least about 200 mg, atleast about 250 mg, at least about 300 mg, at least about 350 mg, atleast about 400 mg, at least about 500 mg, at least about 750 mg, atleast about 1000 mg, and given in one or more doses up to four or moretimes daily.

The form of the pharmaceutical composition of the invention such as apowder, solution, suspension etc. may be suitably selected according tothe type of substance to be administered.

As an administration route, direct inhalation via the mouth using aninhaler is usually administered into the airways and in particular,directly to pulmonary tissue, the active substance contained thereinproduces immediate effects. Furthermore, the composition is formulatedas an immediate release product, as a controlled release product or as acombination of an immediate release product in combination with acontrolled release product so that therapy can begin immediately andcontinue over a period of time to maintain an effective concentration ofactive agent at the site of activity.

The following represents preferred embodiments according to the presentinvention:

Ambroxol Particles for Inhalation

Two main strategies are based around particle size

-   a) Macrophage targeting: In the case of lung infections, where    macrophage delivery of ambroxol would be optimal (such as for TB)    the smallest size of ambroxol particle would be most suited in a    range of 1-2 microns, as this will optimize the phagocytic process.    These 1-2 micron particles could be further coated with a    stabilizing coat, or a coating to enhance macrophage phagocytosis or    recruitment.

These 1-2 micron particles can be delivered as is by nebulization or MDIor in the case of dry powders for inhalation, may be incorporated intoother particles of excipients such as leucine, by spray drying

-   b) Lung epithelial cell targeting: to treat lung epithelial cells,    much smaller ambroxol particles may be needed as these cell primary    route of uptake is endocytosis, not phagocytosis as for macrophages.    Therefore, ambroxol particles in the range of 0.05 to 0.2 microns    are most suited, however these are too small for lung delivery    themselves, and so are incorporated into either nebulized aqueous    aerosols, or in spray dried engineered particles of MMAD 2-5    microns.

EXAMPLES

The Synthesis of compounds according to the present invention proceedsaccording to the chemical scheme which is presented in FIG. 2 hereof. Inessence, 1-hydroxyl-4-amino cyclohexane of set configuration (FIG. 2) isreacted with a dibrominated-amino-benzaldehyde compound which isisotopically labeled as indicated in FIG. 2. One or both of the carbonand hydrogen substituents on the aldehyde aldehyde moiety may beisotopically labeled, depending on the isotopic substitution desired inthe final isotopically labeled ambroxol. In essence, the isotopicallylabeled dibrominated-amino-benzaldehyde compound is condensed with1-hydroxyl-4-amino cyclohexane to produce the isotopically labeled iminecompound, which is subsequently subjected to reduction of the imine(with an optionally isotopically labeled reducing agent) to produce theisotopically labeled ambroxol compound.

Dose Response Assays

Ambroxol dose response assays were conducted and the data generated ispresented in the attached FIGS. 3-6. In brief, bone marrow derivedmacrophages from wild type and ATG5 knockouts were first studied, usinga known inducer of autophagy, pp242 and LC3, puncta was quantified byCellomics as a marker of autophagy, and the dose response of autophagyto ambroxol was determined, using pp242 induction of autophagy as 100%.The dose response of wild type cells is shown in FIG. 3: clearly evenconcentrations as low as 6.25 uM had a positive effect on autophagy (ttest v. control p=0.025). Moreover, it is clear that across the rang ofambroxol concentrations from 0 to 200 uM, higher ambroxol concentrationsresult in higher induction of autophagy.

The inventors next studied the same assay, described above, but in ATG5knockout cells. The data is presented in FIG. 4. Again, evenconcentrations of ambroxol as low as 6.25 uM gave appreciable increasesin autophagy (t test v, control p=0.0008). Again a clear dose responsewas observed. It is noted that the responses for ambroxol reached levelsthat were very much higher than pp242. One interpretation of this isthat the target(s) of ambroxol induced increase in autophagy aredifferent from the known inducer pp242.

The inventors next studied ambroxol induction of TFEB nucleartranslocation as above. The results from the 2 separate experiments areshown in FIGS. 5 and 6, respectively.

A sigmoidal fit of both data sets indicated 50% of maximal autophagyinduced by ambroxol was found at about 50 uM. Again significantincreases were observed at 25 uM. Unlike the LC3 marker of autophagy,ambroxol always gave less effect than pp242.

From the assays performed, it is clear in all cases that increasinglevels of ambroxol will increase autophagic responses.

REFERENCES

-   1. Vergin H, Bishop-Freudling G, Miczka M, Nitsche V, Strobel K,    Matzkies F. 1984. [The pharmacokinetics and bioequivalence of    various dosage forms of ambroxol]. Arzneimittel-Forschung    35:1591-1595.-   2.    ,    ,    ,    , 1977. A Clinico-Pharmacological Study on trans-4-(2-Amino-3,5    dibromobenzylamino) cyclohexanol hydrochloride,    8:25-31.-   3. Ishiguro N, Senda C, Kishimoto W, Sakai K, Funae Y,    Igarashi T. 2000. Identification of CYP3A4 as the predominant    isoform responsible for the metabolism of ambroxol in human liver    microsomes. Xenobiotica 30:71-80.-   4. Anttila S, Hukkanen J, Hakkola J, Stjernvall T, Beaune P, Edwards    R J, Boobis A R, Pelkonen O, Raunio H. 1997. Expression and    localization of CYP3A4 and CYP3A5 in human lung. American journal of    respiratory cell and molecular biology 16:242-249.

1. A compound according to the chemical structure:

or a pharmaceutically acceptable salt, stereoisomer (including anenantiomer or diastereomer), solvate or polymorph thereof.
 2. Thecompound according to claim 1 which is

or a pharmaceutically acceptable salt or enantiomer thereof.
 3. Thecompound according to claim 1 which is

or a pharmaceutically acceptable salt or enantiomer thereof.
 4. Thecompound according to claim 1 which is

or a pharmaceutically acceptable salt thereof.
 5. The compound accordingto claim 1 which is

or a pharmaceutically acceptable salt thereof.
 6. The compound accordingto claim 1 which is

or a pharmaceutically acceptable salt thereof.
 7. The compound accordingto claim 1 which is

or a pharmaceutically acceptable salt thereof.
 8. The compound accordingto claim 1 which is

or a pharmaceutically acceptable salt thereof.
 9. A pharmaceuticalcomposition comprising an effective amount of at least one compoundaccording to claim 1, in combination with a pharmaceutically acceptablecarrier, additive or excipient.
 10. The composition according to claim 9further including an additional bioactive agent.
 11. The compositionaccording to claim 10 wherein said bioactive agent is an autophagymodulator.
 12. The composition according to claim 10 wherein saidadditional bioactive agent is an anti-tuberculosis agent.
 13. Thecomposition according to claim 10 wherein said additional bioactiveagent is a rifamycin.
 14. The composition according to claim 13 whereinsaid rifamycin is rifampin, rifabutin, rifamixin, rifapentine andmixtures thereof.
 15. The composition according to claim 13 whereinrifamycin is rifampin, rifabutin or a mixture thereof.
 16. Thecomposition according to claim 13 wherein said rifamycin is rifampin.17. The composition according to claim 12 wherein said anti-tuberculosisagent is ethambutol, isoniazid, ethionamide, propionamide, pyrazinamide,rifampacin or a mixture thereof.
 18. The composition according to claim11 wherein said autophagy modulator is Astemizole, Chrysophanol,Emetine, Chlorosalicylanilide, Oxiconazole, Sibutramine, Proadifen,Dihydroergotamine tartrate, Terfenadine, Triflupromazine, Amiodarone,Saponin Vinblastine, Tannic acid, Fenticlor, Pizotyline malate,Piperacetazine, Oxyphencyclimine, Glyburide, Hydroxychloroquine,Methotrimeprazine, Mepartricin, Thiamylal Sodium Triclocarban,Diphenidol, Karanjin, Clovanediol diacetate, Nerolidol, Fluoxetine,Helenine, Dehydroabietamide, Dibutyl Phthalate,18-aminoabieta-8,11,13-triene sulfate, Podophyllin acetate, Berbamine,Rotenone, Rubescensin A, Morin, Pyrromycin, Pomiferin, Gardenin A,alpha-mangostin, Avocadene, Butylated hydroxytoluene, Physcion,Tetrandrine, Malathion, Isoliquiritigenin, Clofoctol, Isoreserpine,4,4′-dimethoxydalbergione and 4-methyldaphnetin, benzethonium,niclosamide, monensin, bromperidol, levobunolol, dehydroisoandosterone3-acetate, sertraline, tamoxifen, reserpine, hexachlorophene,dipyridamole, harmaline, prazosin, lidoflazine, thiethylperazine,dextromethorphan, desipramine, mebendazole, canrenone, chlorprothixene,maprotiline, homochlorcyclizine, loperamide, nicardipine,dexfenfluramine, nilvadipine, dosulepin, biperiden, denatonium,etomidate, toremifene, tomoxetine, clorgyline, zotepine, beta-escin,tridihexethyl, ceftazidime, methoxy-6-harmalan, melengestrol,albendazole, rimantadine, chlorpromazine, pergolide, cloperastine,prednicarbate, haloperidol, clotrimazole, nitrofural, iopanoic acid,naftopidil, Methimazole, Trimeprazine, Ethoxyquin, Clocortolone,Doxycycline, Pirlindole mesylate, Doxazosin, Deptropine, Nocodazole,Scopolamine, Oxybenzone, Halcinonide, Oxybutynin, Miconazole,Clomipramine, Cyproheptadine, Doxepin, Dyclonine, Salbutamol, Flavoxate,Amoxapine, Fenofibrate, Pimethixene, a pharmaceutically acceptable saltor alternative pharmaceutically acceptable salt and mixtures thereof.19. The composition according to claim 12 wherein said additionalbioactive agent is an anticancer agent.
 20. The composition according toclaim 15 wherein said anticancer agent is effective for treating lungcancer.
 21. (canceled)
 22. (canceled)
 23. The composition according toclaim 9 in oral, inhalation or pulmonary dosage form.
 24. Thecomposition according to claim 9 in inhalation or pulmonary dosage form.25. The composition according to claim 10, wherein said additionalbioactive agent is at least one additional autophagy modulator,anti-tuberculosis agent or a mixture thereof.
 26. The compositionaccording to claim 19 wherein said compounds is included in saidcomposition as particles having a mean diameter within the range of 0.05microns to 20 microns.
 27. The composition according to claim 22 whereinsaid compound is included in said composition as particles having a meandiameter within the range of 0.05 to 5 microns.
 28. The compositionaccording to claim 19 wherein said compound is included in saidcomposition as particles having a mean diameter within the range ofabout 0.05 to about 0.25 microns.
 29. The composition according to claim19 wherein said compound is included in said composition as particleshaving a mean diameter within the range of about 1 to about 3 microns.30. The composition according to claim 9 in suspension which isdelivered to the lungs of a patient or subject by nebulizing saidsuspension.
 31. The composition according to claim 9 which is deliveredto the lungs of a patient by dry powder inhalation.
 32. The compositionaccording to claim 9 which is delivered to the lungs of a patient bymetered dose inhaler. 33.-61. (canceled)